1. Field of the Invention
The present invention relates to methods for identifying and classifying nucleic acids by means of in situ hybridization of a third-strand probe to a duplex DNA target.
2. Description of Related Art
A list of the references referred to by number herein is found at the end of the Detailed Description of the Invention herein.
Oligodeoxy- and ribo-nucleotide xe2x80x98third strandsxe2x80x99 bind in sequence- and polarity-specific alignment within the major groove of cognate purine-richxe2x80xa2pyrimidine-rich nucleic acid duplex target sequences (1). A third-strand binding code has been elucidated (2) that encompasses triplexes of various structural motifs, of which the best characterized is the pyrlmidine-parallel (Y:Rxe2x80xa2Y) with T:Axe2x80xa2T and C+:Gxe2x80xa2C triplets. The fidelity of third-strand binding to cognate targets of equal and lesser length, targets contained within large DNA molecules, and targets with inverted base pairs, i.e., Rxe2x80xa2Yxe2x86x92Yxe2x80xa2R, is well documented (3). Optimal solution conditions for third-strand binding, i.e., pH, counterionic strength, and temperature, can be manipulated to induce third-strand dissociation or prevent association. While certain triplet mismatches are moderately tolerated within the Y:Rxe2x80xa2Y motif (4), e.g., T:Cxe2x80xa2G and G:Txe2x80xa2A, others are especially destabilizing. Such destabilization provides temperature as a variable to selectively favor, under in vitro and demonstrated in situ conditions, desired triplexes over less perfect ones, as required in the large non-specific duplex DNA background of the human genome, while maintaining essentially constant pH and ionic strength.
The use of third strands for sequence-specific recognition of human genomic DNA has been exploited for the development of potential anti-gene therapeutic agents (5) and artificial endonucleases (6). By virtue of the present invention, this approach is extended to in situ hybridization for the detection and analysis of nucleic acids.
The present invention provides a method for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises:
a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present in the nucleic acid segment; and
b) detecting whether hybridization between the third strand and the target sequence has occurred.